Granzyme b inhibitors

ABSTRACT

The present invention encompasses compounds of Formula (I) and pharmaceutically acceptable salts or hydrates thereof. The compounds are inhibitors of granzyme B and are useful for treating autoimmune and chronic inflammatory diseases. Pharmaceutical compositions and methods of use are also included.

BACKGROUND OF THE INVENTION

Autoimmune diseases are diseases in which a specific immune response toself-molecules occurs, often leading to tissue and organ damage anddysfunction. The diseases can be organ-specific (e.g. Type I diabetesmellitus, thyroiditis, myasthenia gravis, primary biliary cirrhosis) orsystemic in nature (e.g. systemic lupus erythematosus, rheumatoidarthritis, polymyositis, dermatomyositis, Sjogrenfs syndrome,scleroderma, and graft-vs.-host disease).

Apoptosis is a morphologically and biochemically distinct form of celldeath that occurs in many different cell types during a wide range ofphysiologic and pathologic circumstances (reviewed in (Jacobson et al.,1997; Thompson, 1995; White, 1996)). Studies report that specificproteolysis catalyzed by a novel family of cysteine proteases is ofcritical importance in mediating apoptosis (Chinnaiyan and Dixit, 1996a;Martin and Green, 1995; Thornberry and Molineaux, 1995). These proteases(termed caspases), cleave downstream substrates after a consensustetrapeptide sequence ending with aspartic acid. The caspases aresynthesized as inactive precursors that require specific proteolyticcleavage after an aspartic acid residue for activation.

Granzyme B is a serine protease found in the cytoplasmic granules ofcytotoxic T lymphocytes (CTL) and natural killer (NK) cells and has asimilar requirement to caspases for aspartic acid in the substrate P₁position (Odake et al., 1991; Poe et al., 1991). Studies have reportedthat granzyme B plays an important role in inducing apoptotic nuclearchanges in target cells during granule exocytosis induced cytotoxicity(Darmon et al., 1996; Heusel et al., 1994; Sarin et al., 1997; Shrestaet al., 1995; Talanian et al., 1997).

Granzyme B is described as catalyzing the cleavage and activation ofseveral caspases (Chinnaiyan et al., 1996b; Darmon et al., 1995; Duan etal., 1996; Fernandes-Alnemri et al., 1996; Gu et al., 1996; Martin etal., 1996; Muzio et al., 1996; Quan et al., 1996; Sarin et al., 1997;Song et al., 1996a; Srinivasula et al., 1996; Talanian et al., 1997;Wang et al., 1996). Granzyme B also initiates caspase-independentpathways which contribute to target cell death. However, while severalcandidates for these additional pathways exist, they remain largelyundefined (Sarin et al., 1997; Talanian et al., 1997).

One candidate pathway is the direct proteolysis of death substrates bygranzyme B, although efficient non-caspase cellular substrates for thisprotease have not yet been identified. Initial studies have indicatedthat the cleavage of PARP, U1-70 kDa and lamin B observed duringgranzyme B-induced cell death is catalyzed by caspases, rather thandirectly by granzyme B (Darmon et al., 1995; Martinet al., 1996;Talanian et al., 1997), but the effects of granzyme B on other caspasesubstrates in vitro and during granule-induced cytotoxicity have notbeen extensively studied.

The present invention encompasses compounds that are inhibitors ofgranzyme B without inhibiting the caspases. The compounds are thereforeuseful for treating autoimmune and chronic inflammatory disease that maybe specific to CTL-induced cytotoxicity.

SUMMARY OF THE INVENTION

The present invention encompasses compounds of Formula I

and pharmaceutically acceptable salts or hydrates thereof. The compoundsare inhibitors of granzyme B and are useful for treating autoimmune andchronic inflammatory diseases. Pharmaceutical compositions and methodsof use are also included.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses compounds represented by Formula I:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

-   n is 0, 1, or 2;-   R¹ and R² are each independently selected from the group consisting    of: hydrogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₃₋₆cycloalkyl, aryl, HET and    —N(R¹⁰)₂, wherein:    -   (a) said C₁₋₆alkyl, C₁₋₆alkoxy and C₃₋₆cycloalkyl are optionally        substituted with 1-3 substituents independently selected from        the group consisting of halo and hydroxy; and    -   (b) said aryl and BET are optionally substituted with 1-3        substituents independently selected from the group consisting        of: halo, hydroxy and C₁₋₄alkyl, optionally substituted with 1-3        halo groups;-   or R1 and R2 may be joined together with the carbon atom to which    they are attached to form a five or six membered monocyclic ring,    optionally containing 1-3 heteroatoms selected from the group    consisting of: S, O and N(R¹⁰), wherein said ring is optionally    substituted with 1-3 R¹⁰ groups,    with the proviso that R¹ and R² are both not hydrogen;-   each of R³ and R⁷ is independently selected from the group    consisting of: hydrogen and C₁₋₄alkyl, optionally substituted with    1-3 halo groups;-   each of R⁴, R⁵, R⁶ and R⁸ is independently selected from the group    consisting of: hydrogen, halo, hydroxy and C₁₋₄alkyl, optionally    substituted with 1-3 halo groups;-   R⁹ is HET, optionally substituted with 1-3 substituents    independently selected from the group consisting of: halo, hydroxy    and C₁₋₄alkyl, optionally substituted with 1-3 halo groups;-   R¹⁰ is selected from the group consisting of: hydrogen, C₁₋₄alkyl    and —C(O)C₁₋₄alkyl, said —C(O)C₁₋₄alkyl optionally substituted with    N(R¹¹)₂, HET and aryl, said aryl optionally substituted with 1-3    halo groups;-   R¹¹ is selected from hydrogen and C₁₋₄alkyl, optionally substituted    with 1-3 halo groups;-   HET is a 5- to 10-membered aromatic, partially aromatic or    non-aromatic mono- or bicyclic ring, containing 1-4 heteroatoms    selected from O, S and N(R¹²), and optionally substituted with 1-2    oxo groups; and-   R¹² is selected from the group consisting of: hydrogen and    C₁₋₄alkyl, optionally substituted with 1-3 halo groups.

An embodiment of the invention encompasses the compound of Formula Iwherein n is 0.

An embodiment of the invention encompasses the compound of Formula Iwherein n is 1.

An embodiment of the invention encompasses the compound of Formula Iwherein n is 2.

An embodiment of the invention encompasses the compound of Formula Iwherein each of R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is hydrogen.

An embodiment of the invention encompasses the compound of Formula Iwherein BET is selected from the group consisting of: benzimidazolyl,benzofuranyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,benzoxazolyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl,indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl,isothiazolyl, isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl,pyrazinyl, pyrazolyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl,pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thiazolyl,thienyl, triazolyl, tetrazolyl, 1,4-dioxanyl, hexahydroazepinyl,piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, tetrahydrofuranyl, andtetrahydrothienyl, each optionally substituted with 1-2 substituentsindependently selected from the group consisting of: halo, oxo, hydroxyand C₁₋₄alkyl, optionally substituted with 1-3 halo groups.

An embodiment of the invention encompasses the compound of Formula Iwherein R⁹ is selected from the group consisting of: pyridyl, pyrimidyl,pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,oxadiazolyl, isoxazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, andtetrazolyl, each optionally substituted with 1-3 substituentsindependently selected from the group consisting of: halo, hydroxy andC₁₋₄alkyl, optionally substituted with 1-3 halo groups.

An embodiment of the invention encompasses the compound of Formula Iwherein R¹ and R² are each independently selected from the groupconsisting of: C₁₋₆alkyl, C₃₋₆cycloalkyl, phenyl, pyridyl,2-oxopyrrolidine and —N(R¹⁰)₂, wherein:

-   -   (a) said C₁₋₆alkyl and C₃₋₆cycloalkyl optionally substituted        with 1-3 groups independently selected from the group consisting        of halo and hydroxy; and    -   (b) said phenyl, pyridyl and 2-oxopyrrolidine optionally        substituted with 1-3 groups independently selected from the        group consisting of: halo, hydroxy and C₁₋₄alkyl, optionally        substituted with 1-3 halo groups; and

-   R¹⁰ is selected from the group consisting of: hydrogen, C₁₋₄alkyl,    and —C(O)C₁₋₄alkyl, said —C(O)C₁₋₄alkyl optionally substituted with    N(R¹¹)₂, pyrrolidine, piperidine, morpholine, benzothiophene and    phenyl, said phenyl optionally substituted with 1-3 halo groups.

Within this embodiment of the invention is encompassed the compound ofFormula I wherein n is 1. Also within this embodiment is encompassed thecompound of Formula I wherein each of R³, R⁴, R⁵, R⁶, R⁷ and R⁸ ishydrogen. Also within this embodiment is encompassed the compound ofFormula I wherein R⁹ is selected from the group consisting of: pyridyl,pyrimidyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, thiazolyl,oxazolyl, oxadiazolyl, isoxazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, andtetrazolyl, each optionally substituted with 1-3 substituentsindependently selected from the group consisting of: halo, hydroxy andC₁₋₄alkyl, optionally substituted with 1-3 halo groups.

Another embodiment of the invention encompasses a compound of FormulaII:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

-   R⁹ is selected from the group consisting of: benzimidazolyl,    benzofuranyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,    benzoxazolyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl,    indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl,    isothiazolyl, isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl,    pyrazinyl, pyrazolyl, pyridopyridinyl, pyridazinyl, pyridyl,    pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,    thiadiazolyl, thiazolyl, thienyl, triazolyl, tetrazolyl,    1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,    pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl,    dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl,    dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,    dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,    dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl,    dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl,    dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl,    dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl,    tetrahydrofuranyl, and tetrahydrothienyl.

Within this embodiment is encompassed the compound of Formula I whereinR⁹ is selected from the group consisting of: pyridyl, pyrimidyl,pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,oxadiazolyl, isoxazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, andtetrazolyl.

Another embodiment of the invention encompasses a pharmaceuticalcomposition comprising a compound of Formula I in combination with apharmaceutically acceptable carrier.

Another embodiment of the invention encompasses a method of treating animmunoregulatory abnormality in a mammalian patient in need of suchtreatment comprising administering to said patient a compound inaccordance with claim 1 in an amount that is effective for treating saidimmunoregulatory abnormality.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is an autoimmune or chronicinflammatory disease selected from the group consisting of: systemiclupus erythematosis, chronic rheumatoid arthritis, type I diabetesmellitus, inflammatory bowel disease, biliary cirrhosis, uveitis,multiple sclerosis, Crohn's disease, ulcerative colitis, bullouspemphigoid, sarcoidosis, psoriasis, autoimmune myositis, Wegener'sgranulomatosis, ichthyosis, Graves ophthalmopathy, asthma, schlerodermaand Sjogren's syndrome.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is bone marrow or organ transplantrejection or graft-versus-host disease.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is selected from the group consistingof: transplantation of organs or tissue, graft-versus-host diseasesbrought about by transplantation, autoimmune syndromes includingrheumatoid arthritis, systemic lupus erythematosus, Hashimoto'sthyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes,uveitis, posterior uveitis, allergic encephalomyelitis,glomerulonephritis, post-infectious autoimmune diseases includingrheumatic fever and post-infectious glomerulonephritis, inflammatory andhyperproliferative skin diseases, psoriasis, atopic dermatitis, contactdermatitis, eczematous dermatitis, seborrhoeic dermatitis, lichenplanus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria,angioedemas, vasculitis, erythema, cutaneous eosinophilia, lupuserythematosus, acne, alopecia areata, keratoconjunctivitis, vernalconjunctivitis, uveitis associated with Behcet's disease, keratitis,herpetic keratitis, conical cornea, dystrophia epithelialis corneae,corneal leukoma, ocular pemphigus, Mooren's ulcer, scleritis, Graves'opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollenallergies, reversible obstructive airway disease, bronchial asthma,allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma,chronic or inveterate asthma, late asthma and airwayhyper-responsiveness, bronchitis, gastric ulcers, vascular damage causedby ischemic diseases and thrombosis, ischemic bowel diseases,inflammatory bowel diseases, necrotizing enterocolitis, intestinallesions associated with thermal burns, coeliac diseases, proctitis,eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerativecolitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischemic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, chronic bacterialinfection, malignancy of lymphoid origin and acute and chroniclymphocytic leukemias and lymphomas.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is multiple sclerosis.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is rheumatoid arthritis.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is systemic lupus erythematosus.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is psoriasis.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is rejection of transplanted organ ortissue.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is inflammatory bowel disease.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is a malignancy of lymphoid origin.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is acute and chronic lymphocyticleukemias and lymphomas.

Another embodiment of the invention encompasses the above method whereinthe immunoregulatory abnormality is selected from the group consistingof: schleroderma, autoimmune myositis, Sjogren's syndrome and type Idiabetes.

Another embodiment of the invention encompasses a method of suppressingthe immune system in a mammalian patient in need of immunosuppressioncomprising administering to said patient an immunosuppressing effectiveamount of a compound of Formula I.

Another embodiment of the invention encompasses a pharmaceuticalcomposition comprising a compound which inhibits granzme B and does notsubstantially inhibit any caspase protease in combination with apharmaceutically acceptable carrier.

Another embodiment of the invention encompasses a pharmaceuticalcomposition comprising a compound which possesses a Ki of 500 nM or lessfor inhibiting granzyme B and possesses a Ki of 10,000 nM or more forinhibiting each of caspase-1 to caspase-13 in combination with apharmaceutically acceptable carrier.

Another embodiment of the invention encompasses a method of treating animmunoregulatory abnormality in a mammalian patient in need of suchtreatment comprising administering to said patient a compound whichinhibits granzme B and does not substantially inhibit any caspaseprotease in an amount that is effective for treating saidimmunoregulatory abnormality.

Another embodiment of the invention encompasses method of treating animmunoregulatory abnormality in a mammalian patient in need of suchtreatment comprising administering to said patient a compound whichpossesses a Ki of 500 nM or less for inhibiting granzyme B and possessesa Ki of 10,000 nM or more for inhibiting each of caspase-1 to caspase-13in an amount that is effective for treating said immunoregulatoryabnormality.

For purposes of this Specification, references to the activity of thecompounds are as measured in the assays disclosed herein.

Exemplifying the invention are compounds of the following table: TABLE 1

EXAMPLE # A B 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

The invention is described using the following definitions unlessotherwise indicated.

The term “halogen” or “halo” includes F, Cl, Br, and I.

The term “alkyl” means linear or branched structures and combinationsthereof, having the indicated number of carbon atoms. Thus, for example,C₁₋₆alkyl includes methyl, ethyl, propyl, 2-propyl, s- and t-butyl,butyl, pentyl, hexyl, 1,1-dimethylethyl, cyclopropyl, cyclobutylcyclopentyl and cyclohexyl.

The term “alkoxy” means alkoxy groups of a straight, branched or cyclicconfiguration having the indicated number of carbon atoms. C₁₋₆alkoxy,for example, includes methoxy, ethoxy, propoxy, isopropoxy, and thelike.

The term “alkylthio” means alkylthio groups having the indicated numberof carbon atoms of a straight, branched or cyclic configuration.C₁₋₆alkylthio, for example, includes methylthio, propylthio,isopropylthio, and the like.

The term “alkenyl” means linear or branched structures and combinationsthereof, of the indicated number of carbon atoms, having at least onecarbon-to-carbon double bond, wherein hydrogen may be replaced by anadditional carbon-to-carbon double bond. C₂₋₆alkenyl, for example,includes ethenyl, propenyl, 1-methylethenyl, butenyl and the like.

The term “alkynyl” means linear or branched structures and combinationsthereof, of the indicated number of carbon atoms, having at least onecarbon-to-carbon triple bond. C₃₋₆alkynyl, for example, includes,propenyl, 1-methylethenyl, butenyl and the like.

The term “cycloalkyl” means mono-, bi- or tri-cyclic structures,optionally combined with linear or branched structures, the indicatednumber of carbon atoms. Examples of cycloalkyl groups includecyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl,2-ethyl-1-bicyclo[4.4.0]decyl, and the like.

The term “aryl” is defined as a mono- or bi-cyclic aromatic ring systemand includes, for example, phenyl, naphthyl, and the like.

The term “aralkyl” means an alkyl group as defined above of 1 to 6carbon atoms with an aryl group as defined above substituted for one ofthe alkyl hydrogen atoms, for example, benzyl and the like.

The term “aryloxy” means an aryl group as defined above attached to amolecule by an oxygen atom (aryl-O) and includes, for example, phenoxy,naphthoxy and the like.

The term “aralkoxy” means an aralkyl group as defined above attached toa molecule by an oxygen atom (aralkyl-O) and includes, for example,benzyloxy, and the like.

The term “arylthio” is defined as an aryl group as defined aboveattached to a molecule by an sulfur atom (aryl-S) and includes, forexample, thiophenyoxy, thionaphthoxy and the like.

The term “aroyl” means an aryl group as defined above attached to amolecule by an carbonyl group (aryl-C(O)—) and includes, for example,benzoyl, naphthoyl and the like.

The term “aroyloxy” means an aroyl group as defined above attached to amolecule by an oxygen atom (aroyl-O) and includes, for example,benzoyloxy or benzoxy, naphthoyloxy and the like.

The term “HET” is defined as a 5- to 10-membered aromatic, partiallyaromatic or non-aromatic mono- or bicyclic ring, containing 1-4heteroatoms selected from O, S and N, and optionally substituted with1-2 oxo groups. Preferably, “HET” is a 5- or 6-membered aromatic ornon-aromatic monocyclic ring containing 1-5 heteroatoms selected from O,S and N, for example, pyridine, pyrimidine, pyridazine, furan,thiophene, thiazole, oxazole, isooxazole and the like, or heterocycle isa 9- or 10-membered aromatic or partially aromatic bicyclic ringcontaining 1-5 heteroatoms selected from O, S, and N, for example,benzofuran, benzothiophene, indole, pyranopyrrole, benzopyran,quionoline, benzocyclohexyl, naphtyridine and the like. “HET” alsoincludes the following: benzimidazolyl, benzofuranyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl,indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl,pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, tetrazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl,piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl.

The term “treating” encompasses not only treating a patient to relievethe patient of the signs and symptoms of the disease or condition butalso prophylactically treating an asymptomatic patient to prevent theonset or progression of the disease or condition. The term “amounteffective for treating” is intended to mean that amount of a drug orpharmaceutical agent that will elicit the biological or medical responseof a tissue, a system, animal or human that is being sought by aresearcher, veterinarian, medical doctor or other clinician. The termalso encompasses the amount of a pharmaceutical drug that will preventor reduce the risk of occurrence of the biological or medical event thatis sought to be prevented in a tissue, a system, animal or human by aresearcher, veterinarian, medical doctor or other clinician.

For purposes of this specification, the following abbreviations have theindicated meanings: AcOH = acetic acid Alloc = allyloxycarbonyl APCI =atmospheric pressure chemical ionization BOC = t-butyloxycarbonyl CBZ =carbobenzoxy DCC = 1,3-dicyclohexylcarbodiimide DIBAL = diisobutylaluminum hydride DIEA = N,N-diisoproylethylamine DMAP =4-(dimethylamino)pyridine DMF = dimethyl formamide DTT = dithiothreitolEDCI = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDTA= ethylenediaminetetraacetic acid, tetrasodium salt hydrate ESI =electrospray ionization FAB = fast atom bombardment FMOC =9-fluorenylmethoxycarbonyl HMPA = hexamethylphosphoramide HATU =O-(7-Azabenzotriazol-1-yl)N,N,N′,N′- tetramethyluroniumhexafluorophosphate HOBT = 1-hydroxybenzotriazole HRMS = high resolutionmass spectrometry ICl = iodine monochloride IBCF = isobutylchloroformate KHMDS = potassium hexamethyldisilazane LDA = lithiumdiisopropylamide MCPBA = metachloroperbenzoic acid Ms = methanesulfonyl= mesyl MsO = methanesulfonate = mesylate NBS = N-bromosuccinimide NMM =4-methylmorpholine PCC = pyridinium chlorochromate PDC = pyridiniumdichromate Ph = phenyl PPTS = pyridinium p-toluene sulfonate pTSA =p-toluene sulfonic acid r.t. = room temperature rac. = racemic TFA =trifluoroacetate TfO = trifluoromethanesulfonate = triflate TLC = thinlayer chromatography

Alkyl group abbreviations: Me = methyl Et = ethyl n-Pr = normal propyli-Pr = isopropyl n-Bu = normal butyl i-Bu = isobutyl s-Bu = secondarybutyl t-Bu = tertiary butyl

The compounds described herein are intended to include salts,enantiomers, esters and hydrates, in pure form and as a mixture thereof.Also, when a nitrogen atom appears, it is understood sufficient hydrogenatoms are present to satisfy the valency of the nitrogen atom.

While chiral structures are shown below, by substituting into thesynthesis schemes an enantiomer other than the one shown, or bysubstituting into the schemes a mixture of enantiomers, a differentisomer or a racemic mixture can be achieved. Thus, all such isomers andmixtures are included in the present invention.

The compounds described typically contain asymmetric centers and maythus give rise to diastereomers and optical isomers. The presentinvention is meant to comprehend such possible diastereomers as well astheir racemic and resolved, enantiomerically pure forms andpharmaceutically acceptable salts thereof.

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and Zgeometric isomers.

The pharmaceutical compositions of the present invention comprise acompound of Formula I as an active ingredient or a pharmaceuticallyacceptable salt thereof in combination with a pharmaceuticallyacceptable carrier, and optionally other therapeutic ingredients. Theterm “pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable bases including inorganic bases and organicbases. Representative salts derived from inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic salts, manganous, ammonium, potassium, sodium, zincand the like. Particularly preferred are the calcium, magnesium,potassium, and sodium salts. Representative salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines, and basic ion exchangeresins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like.

When the compound of the present invention is basic, salts may beprepared from pharmaceutically acceptable non-toxic acids, includinginorganic and organic acids. Examples of such acids include acetic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic,fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic,lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicacid, and the like. Particularly preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.

In the discussion of methods of treatment that follows, reference to thecompounds of Formula I are meant to also include the pharmaceuticallyacceptable salts.

By virtue of their Granzyme B inhibiting activity, the compounds of thepresent invention are useful for treating or preventing automimmune orchronic inflammatory diseases. The compounds of the present inventionare useful to suppress the immune system in instances whereimmunosuppression is in order, such as in bone marrow, organ ortransplant rejection, autoimmune and chronic inflammatory diseases,including systemic lupus erythematosis, chronic rheumatoid arthritis,type I diabetes mellitus, inflammatory bowel disease, biliary cirrhosis,uveitis, multiple sclerosis, Crohn's disease, ulcerative colitis,bullous pemphigoid, sarcoidosis, psoriasis, autoimmune myositis,Wegener's granulomatosis, ichthyosis, Graves ophthalmopathy, asthma,schleroderma and Sjogren's syndrome.

More particularly, the compounds of the present invention are useful totreat or prevent a disease or disorder selected from the groupconsisting of: transplantation of organs or tissue, graft-versus-hostdiseases brought about by transplantation, autoimmune syndromesincluding rheumatoid arthritis, systemic lupus erythematosus,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type Idiabetes, uveitis, posterior uveitis, allergic encephalomyelitis,glomerulonephritis, post-infectious autoimmune diseases includingrheumatic fever and post-infectious glomerulonephritis, inflammatory andhyperproliferative skin diseases, psoriasis, atopic dermatitis, contactdermatitis, eczematous dermatitis, seborrhoeic dermatitis, lichenplanus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria,angioedemas, vasculitis, erythema, cutaneous eosinophilia, lupuserythematosus, acne, alopecia areata, keratoconjunctivitis, vernalconjunctivitis, uveitis associated with Behcet's disease, keratitis,herpetic keratitis, conical cornea, dystrophia epithelialis corneae,corneal leukoma, ocular pemphigus, Mooren's ulcer, scleritis, Graves'opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollenallergies, reversible obstructive airway disease, bronchial asthma,allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma,chronic or inveterate asthma, late asthma and airwayhyper-responsiveness, bronchitis, gastric ulcers, vascular damage causedby ischemic diseases and thrombosis, ischemic bowel diseases,inflammatory bowel diseases, necrotizing enterocolitis, intestinallesions associated with thermal burns, coeliac diseases, proctitis,eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerativecolitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischemic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, and chronic bacterialinfection.

The magnitude of therapeutic dose of a compound of Formula I will, ofcourse, vary with the nature of the severity of the condition to betreated and with the particular compound of Formula I and its route ofadministration and vary upon the clinician's judgement. It will alsovary according to the age, weight and response of the individualpatient. An effective dosage amount of the active component can thus bedetermined by the clinician after a consideration of all the criteriaand using is best judgement on the patient's behalf. A representativedose will range from 0.001 mpk/d to about 100 mpk/d.

An ophthalmic preparations for ocular administration comprising 0.001-1%by weight solutions or suspensions of the compounds of Formula I in anacceptable ophthalmic formulation may be used.

Any suitable route of administration may be employed for providing aneffective dosage of a compound of the present invention. For example,oral, parenteral and topical may be employed. Dosage forms includetablets, troches, dispersions, suspensions, solutions, capsules, creams,ointments, aerosols, and the like.

The compositions include compositions suitable for oral, parenteral andocular (ophthalmic). They may be conveniently presented in unit dosageform and prepared by any of the methods well-known in the art ofpharmacy.

In practical use, the compounds of Formula I can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration. In preparing the compositionsfor oral dosage form, any of the usual pharmaceutical media may beemployed, such as, for example, water, alcohols, oils, flavoring agents,preservatives, coloring agents and the like in the case of oral liquidpreparations, such as, for example, suspensions, elixirs and solutions;or carriers such as starches, sugars, microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like in the case or oral solid preparations such as, forexample, powders, capsules and tablets, with the solid oral preparationsbeing preferred over the liquid preparations. Because of their ease ofadministration, tablets and capsules represent the most advantageousoral dosage unit form in which case solid pharmaceutical carriers areobviously employed. If desired, tablets may be coated by standardaqueous or nonaqueous techniques.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient, as a powder or granules or as a solution or a suspension inan aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or awater-in-oil emulsion. Such compositions may be prepared by any of themethods of pharmacy but all methods include the step of bringing intoactive ingredient with the carrier which constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Forexample, a tablet may be prepared by compression or molding, optionallywith one or more accessory ingredients. Compressed tablets may beprepared by compressing in a suitable machine, the active ingredient ina free-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent. Forexample, each dosage unit may contain from about 0.01 mg to about 1.0 gof the active ingredient.

Methods of Synthesis

The compounds of the present invention are prepared using the generalprocedures described below:

Commercially available(2S,5S)-5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylicacid can be converted to the benzyl ester by reaction with benzylbromide in the presence of a suitable base such asN,N-diisopropylethylamine or the like. Removal of the FMOC protectinggroup can be carried out with diethyl amine and the free amine can besubsequently coupled with a carboxylic acid using standard peptidecoupling conditions. Removal of the benzyl protecting group can beconducted by catalytic hydrogenation with palladium or alternatively byhydrolysis with a suitable base such as lithium hydroxide. Coupling withthe requisite amine can be accomplished with standard peptide couplingconditions such as with EDC/HOBt to afford the desired compounds.

Methods for preparing the compounds of this invention are furtherillustrated in the following examples. Alternative routes will be easilydiscernible to practitioners in the field.

EXAMPLE 1

(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-tetraazol-5-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

Step A: Benzyl(2S,5S)-5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate

A solution of(2S,5S)-5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylicacid (1.5 grams, 3.2 mmol), N,N-diisopropylethyl amine (0.73 mL, 4.2mmol) and benzyl bromide (0.46 mL, 3.8 mmol) were stirred in 4 mL DMFfor 12 h. The mixture was diluted with EtOAc and washed with water (3×)and sat'd NaCl. The organic layer was dried over sodium sulfate andconcentrated. Flash chromatography (3/1 hexanes/EtOAc) gave 1.6 grams(90%) product. ¹H NMR (500 MHz, CDCl₃) δ, 7.81 (d, 2H), 7.65 (d, 2H),7.31-7.44 (6H), 7.12-7.21 (3H), 6.28 (d, 1H), 5.39 (d, 1H), 5.21 (dd,2H), 4.42 (m, 2H), 4.37 (m, 1H), 4.28 (m, 1H, 3.53 (m, 2H), 3.38 (m,1H), 3.17 (m, 2H), 2.41 (m, 1H), 2.18 (m, 1H).

Step B: Benzyl(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate

A solution of benzyl(2S,5S)-5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate(1.6 grams, 2.8 mmol, from example 1 Step A) in 20 mL 1/1diethylamine/dichloromethane was stirred for 2 h. The mixture wasevaporated to dryness under vacuum then redissolved in 15 mLdichloromethane. Acetyl isoleucine (744 mg, 4.3 mmol), EDC (660 mg, 3.4mmol) and HOBt (775 mg, 5.7 mmol) were added and the mixture was stirredfor 10 h. The mixture was diluted with EtOAc and washed with 1 M HCl,sat'd sodium bicarbonate (2×) and sat'd NaCl. The organic layer wasdried and concentrated. Flash chromatography (1/1 dichloromethane/ether)gave 1.2 grams (85%) of product. LC-MS (ESI) calc (M+H) 492.2, found.492.3.

Step C:(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylic acid

A solution of benzyl(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate(1.2 grams, 2.4 mmol, from example 1 Step B) in 40 mL methanol wasstirred over palladium on carbon (260 mg, 10% wt., 0.24 mmol) under anatmosphere of hydrogen for 2 h. The mixture was filtered andconcentrated to afford 0.93 grams (95%) of product. ¹ H NMR (500 MHz,DMSO)

, 8.21 (d, 1H), 7.93 (d, 1H), 7.15 (d, 1H), 7.08 (d, 1H), 6.95 (dd, 1H),5.05 (d, 1H), 4.35 (m, 1H), 4.25 (t, 1H), 3.5 (dd, 1H), 3.2-3.4 (bs,1H), 32.97-3.2 (m, 3H), 1.97-2.17 (m, 2H), 1.88 (s, 3H), 1.7-1.8 (m,1H), 1.4-1.48 m, 1H), 1.08-1.17 (m, 1H), 0.9 (d, 3H), 0.82 (t, 3H).

Step D:(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-N-(cyanomethyl)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

A mixture of(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylic acid (100 mg, 0.25 mmol, from example 1Step C), aminoacetonitrile (17 mg, 0.3 mmol), EDC (72 mg, 0.357 mmol)and HOBt (68 mg, 0.5 mmol) were stirred together in 1 mL DMSO. After 1 hthe mixture was purified by semi-prep HPLC (Column: YMC Pro-pack C18 5μ,20×100 mm, gradient: 20%→80% acetonitrile/water with 0.1% TFA, 20mL/min) to afford 86 mg (78%) of product. ¹H NMR (500 MHz, DMSO)

, 8.68 (d, 1H), 8.25 (d, 1H), 7.93 (d, 2H), 7.11 (d, 1H), 7.05 (d, 1H),6.98 (dd, 1H), 5.02 (d, 1H), 4.37 (m, 1H), 4.23 (t, 1H), 4.12 (m, 2H),3.44 (dd, 1H), 3.1 (m, 2H), 2.89 (d, 1H), 2.02-2.14 (m, 2H), 1.84 (s,3H), 1.73 (m, 1H), 1.43 (m, 1H), 1.13 (m, 1H), 0.9 (d, 3H), 0.83 (t,3H).

Step E:(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-tetraazol-5-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

A mixture of(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-N-(cyanomethyl)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide(27 mg, 0.06 mmol, from example 1 Step D), sodium azide (20 mg, 0.31mmol) and triethylamine hydrochloride (51 mg, 0.37 ml) in 1 mL DMF washeated to 115° C. for 6 h. The product was isolated by semi-prep HPLC(Column: YMC Pro-pack C18 5 μ, 20×100 mm, gradient: 10%→70%acetonitrile/water with 0.1% TFA, 20 mL/min). ¹H NMR (500 MHz, DMSO)

, 8.74 (m, 1H), 8.21 (d, 1H), 7.92 (d, 1H), 7.08 (d, 1H), 7.04 (d, 1H),6.95 (dd, 1H), 5.04 (d, 1H), 4.52 (ABX, 2H), 4.32 (m, 1H), 4.20 (t, 1H),3.42 (dd, 1H), 3.05 (m, 2H), 2.98 (d, 1H), 2.03 (m, 2H), 1.84 (s, 3H),1.73 (m, 1H), 1.42 (m, 1H), 1.11 (m, 1H), 0.86 (d, 3H), 0.81 (t, 3H).LC-MS (ESI) calc (M+H) 483.2, found. 483.3.

EXAMPLE 2

(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

Step A: 1-(1H-1,2,3-triazol-4-yl)methylamine

Sodium azide (11.5 grams, 176 mmol), and propargyl bromide (27.7 grams,80% wt solution in toluene, 186 mmol) were stirred in a mixture of 200mL dioxane and 150 mL water for 10 h. The aqueous layer was separatedand the organic layer was transferred to a pressure reactor.Concentrated ammonium hydroxide (200 mL) was added and the mixture washeated to 65° C. After 15 h the mixture was cooled and concentrated.Crystallization from dioxane/water afforded 5.2 grams (30%) of pureproduct. ¹H NMR (500 MHz, DMSO)

, 7.62 (s, 1H), 5.1-5.5 (bs, 3H), 3.79 (s, 2H).

Step B:(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

The compound was prepared according to the procedure in example 1 step Dfrom(2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylicacid (100 mg, 0.25 mmol, from example 1 Step C) and1-(1H-1,2,3-triazol-4-yl)methylamine (50 mg, 0.5 mmol, from example 2Step A). The product was purified by semi-prep HPLC (Column: YMCPro-pack C18 5μ, 20×100 mm, gradient: 20%→40% acetonitrile/water with0.1% TFA, 20 mL/min) to give 63 mg (52%). ¹H NMR (500 MHz, DMSO)

, 8.47 (m, 1H), 8.22 (d, 1H), 7.91 (d, 1H), 7.6 (bs, 1H), 7.08 (d, 1H),7.04 (d, 1H), 6.95 (dd, 1H), 5.01 (d, 1H), 4.32 (m, 3H), 4.21 (t, 1H),3.42 (dd, 1H), 3.07 (m, 2H), 2.95 (d, 1H), 2.03 (m, 2H), 1.84 (s, 3H),1.72 (m, 1H), 1.42 (m, 1H), 1.11 (m, 1H), 0.87 (d, 3H), 0.81 (t, 3H).LC-MS (ESI) calc (M+H) 482.2, found. 482.3

EXAMPLE 3

(2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

Step A: Benzyl pyridin-2-ylacetate

2-pyridyl acetic acid hydrochloride (8 grams, 46 mmol), benzyl alcohol(19 mL, 184 mmol), EDC (13 grams, 69 mmol), N,N-diisopropylethyl amine(8 mL, 46 mmol) and DMAP (560 mg, 4.6 mmol) were combined in 150 mLdichloromethane and the mixture was stirred overnight. The mixture wasdiluted with EtOAc and extracted with 2M HCl (2×). The combined aqueouslayers were neutralizes with solid sodium bicarbonate and extracted withEtOAc. The organic portion was dried with sodium sulfate andconcentrated. Flash chromatography (2/1 hexanes/EtOAc) gave 8.6 grams(86%) of product. ¹H NMR (500 MHz, CDCl₃)

, 8.59 (d, 1H), 7.66 (dd, 1H), 7.3-7.4 (m, 6H), 7.2 (m, 1H), 5.19 (s,2H), 3.96 (s, 2H).

Step B: Benzyl 3-methyl-2-pyridin-2-ylbutanoate

A solution of lithium hexamethyl disilazide (5.5 mL, 1.0 M in THF, 5.5mmol) in and additional 15 mL THF was cooled to −78° C. under a nitrogenatmosphere. Benzyl pyridin-2-ylacetate (1.1 g, 5 mmol, from example 3Step A) was added and the resulting mixture was stirred for 30 min.2-Iodo propane (0.55 mL, 5.5 mmol) was added and the mixture was warmedto room temperature then stirred for 12 h. The reaction mixture waspoured into sat'd ammonium chloride and extracted with EtOAc. Theorganic portion was dried over sodium sulfate and concentrated. Flashchromatography (5/1 hexane/EtOAc) gave 0.9 grams (67%) of product.

Step C: 3-Methyl-2-pyridin-2-ylbutanoic acid

A solution of benzyl 3-methyl-2-pyridin-2-ylbutanoate (0.9 grams, 3.3mmol, from example 3 Step B) in 15 mL ethanol was stirred with palladiumon carbon (350 mg, 0.33 mmol, 10% wt.) under 1 atmosphere of hydrogen.After 1.5 h the mixture was filtered through celite and concentrated togive 550 mg (93%) product. The product must be used immediately as itspontaneously looses CO₂. ¹H NMR (500 MHz, CD₃OD)

, 848 (d, 1H), 7.82 (dd, 1H), 7.55 (d, 1H), 7.34 (m, 1H), 3.42 (d, 1H),2.42 (m, 1H), 1.1 (d, 3H), 0.73 (d, 3H).

Step D: Benzyl(2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate

The title compound was prepared from 3-Methyl-2-pyridin-2-ylbutanoicacid (1.5 grams, 8.5 mmol, from example 3 Step C) and benzyl(2S,5S)-5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate(3.05 grams, 5.5 mmol, from example 1 Step A). Flash chromatography (5/1hexanes/EtOAc) gave 1.7 grams (67%) of product. The desired diastereomer(faster eluting isomer) was isolated by chiral semi-prep HPLC (350 gramschiral-pak AD stationary phase eluting with isopropanol).

Step E:(2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylicacid

Benzyl(2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate(550 mg, 1.1 mmol from example 3 Step D) was hydrogenated using theprocedure described in example 1 Step C to afford 410 mg (92%) product.¹H NMR (500 MHz, DMSO)

, 8.67 (d, 1H), 8.54 (d, 1H), 7.72 (dd, 1H), 7.43 (d, 1H), 7.25 (m, 1H),7.1 (d, 1H), 7.03 (d, 1H), 6.96 (m, 1H), 5.03 (d, 1H), 4.37 (t, 1H),3.48 (m, 1H), 2.4 (d, 1H), 2.98-3.15 (m, 3H), 2.38 (m, 1H), 1.95 (m,2H), 1.05 (d, 3H), 0.65 (d, 3H).

Step F:(2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

The title compound was prepared from(2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylicacid (31 mg, 0.075 mmol, from example 3 Step E) and1-(1H-1,2,3-triazol-4-yl)methylamine (15 mg, 0.15 mmol, from example 2Step A) using the procedure described in example 2 Step B). ¹H NMR (500MHz, DMSO)

, 8.64 (d, 1H), 8.47-8.51 (m, 2H), 7.70 (dd, 1H), 7.41 (d, 1H), 7.23 (m,1H), 7.06 (d, 1H), 7.00 (d, 1H),6.93 (dd, 1H), 5.0 (d, 1H), 4.23-4.35(h, 3H), 3.35-3.55 (m, 2H), 3.29 (m, 4H), 2.98-3.05 (m, 2H), 2.89 (d,H), 2.34 (m, 1H), 1.94-2.04 (m, 2H), 1.02 (d, 3H), 0.62 (d, 2H). LC-MS(ESI) calc (M+H) 487.2, found. 487.3

EXAMPLE 4

(2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

Step A: N-(phenylacetyl)-L-isoleucine

A solution of isoleucine benzyl ester p-toluenesulfonate (3.9 grams, 10mmol), phenyl acetic acid (1.4 grams, 10 mmol), EDC (2.3 grams, 12mmol), HOBt (2.0 grams, 15 mmol) and N,N-diisopropyl ethylamine (1.9 mL,11 mmol) were combined in 50 mL dichloromethane and the mixture wasstirred for 3 h. The reaction mixture was diluted with EtOAc and washedwith sat'd sodium bicarbonate (3×), water and sat'd NaCl. The organiclayer was dried over sodium sulfate and concentrated. Flashchromatography (3/1 hexanes/EtOAc) gave 3.4 grams of product. The esterwas dissolved in 50 mL methanol and stirred over palladium on carbon(1.0 grams, 1 mmol, 10% wt.) under a hydrogen atmosphere. After 2 h themixture was filtered through celite and concentrated to afford 1.9 grams(75%) of the desired acid.

Step B:(2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylicacid

The title compound was prepared from N-(phenylacetyl)-L-isoleucine (100mg, 0.40 mmol, from example 4 Step A) and benzyl(2S,5S)-5-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylate(150 mg, 0.27 mmol, from example 1 Step A) using the proceduresdescribed in example 1 Steps B and C.

Step C:(2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide

The title compound was prepared from(2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylicacid (36 mg, 0.075 mmol, from example 4 Step B) and1-(1H-1,2,3-triazol-4-yl)methylamine (15 mg, 0.15 mmol, from example 2Step A) using the procedure described in example 2 Step B). ¹H NMR (500MHz, DMSO)

, 8.47 (t, 1H), 8.28 (d, 1H), 8.11 (d, 1H), 7.25 (m, 4H), 7.19 (m, 1H),7.07 (d, 1H), 7.03 (d, 1H), 6.95 (dd, 1H) 5.01 (d, 1H), 4.20-4.36 (m,4H), 3.48 (AB, 2H), 3.43 (m 1H), 3.05 (m, 2H), 2.89 (d, 2H), 2.03 (m,2H), 1.75 (m, 1H), 1.42 (m, 1H), 1.08 (m, 1H), 0.84 (d, 3H), 0.78 (t,3H). LC-MS (ESI) calc (M+H) 558.2, found. 558.3

EXAMPLES 5 TO 33

Examples 5 to 33 shown in table 2 were prepared using proceduresanalogous to those in examples 1 to 4 TABLE 2

EXAMPLE # A B LC-MS (ESI) M + H 5

482.3 6

481.3 7

481.3 8

481.3 9

498.2 10

482.3 11

498.2 12

482.3 13

498.2 14

493.3 15

493.3 16

492.3 17

492.3 18

492.3 19

532.3 20

548.3 21

489.4 22

489.4 23

559.3 24

595.3 25

526.4 26

615.3 27

525.3 28

614.3 29

512.1 30

508.3 31

437.2 32

466.2 33

494.3Assays for Determining Biological Activity

The Granzyme B inhibitory activity of the compounds of the presentinvention can be evaluated using the following procedures:

Granzyme B Production.

Plasmids pMelBac and the TA Cloning Kit (pCR2.1) were purchased fromInvitrogen (Carlsbad, Calif.). The Bac-to-Bac Baculovirus ExpressionSystem and all cell culture supplies were from Life Technologies(Gaithersburg, Md.). The human granzyme B protease has been identifiedand is known in the art. See, for example, Poe, et al., J. Biol. Chem.266, 98-103. A vector was generated for secreted protein expression inthe Bac-to-Bac system. The honeybee melittin secretion signal wasamplified from pMelBac by PCR with primers 5′-GTGT AGA TCT ATG AAA TTCTTA GTC AAC G-3′ and 5′-TTC AGC AGA GTC GAC TCC AAG-3′. The amplifiedproduct contained a BglII site upstream of the melittin secretion signaland spanned the multiple cloning site of the vector. The fragment wasdigested with BglII and EcoRI and subcloned into BamHI and EcoRIdigested pFastBac. The resulting vector was designated pSecBac.Activatable tagged human granzyme B was cloned into pSecBac in twosequential steps. Firstly, mature granzyme B was amplified by PCR withprimers 5′-GGATCC ATC GAA GGT CGT ATC ATC GGAGGACATGAGGCC-3′ and 5′-AAGCTT TTA GTA GCG CTT CAT GGT CTT CTT TAT CC-3′ and cloned into pCR2.1. Inthe sense primer, the bold type sequence coding for the Factor Xarecognition site, Ile-Glu-Gly-Arg, was inserted upstream of isoleucine21 (referred to as ILE 16 in this paper using the numbering scheme forthis family of serine proteases is traditionally based on the zymogen,chymotrypsinogen), the first amino acid of mature granzyme B. Secondly,hexa-histidine tagged, activatable granzyme B was amplified from thegranzyme B/pCR2.1 clone by PCR using primers 5′-GGA AGA TCT CAT CAT CATCAT CAT CAT GGA TCC ATC GAA GGT CGT ATC-3′ and 5′-CCT GAA TTC TTA GTAGCG TIT CAT GGT CTT CTT TAT CC-3′. The amplified product contained aBali site upstream of the hex-histidine tag and contained the Factor Xarecognition sequence. This fragment was digested with Bali and ECOR andsubcloned into BamHI and EcoRI digested pSecBac. The completed vectorgenerated a recombinant baculovirus in Gibco BRL's Bac-to-Bac system,which would produce a secreted, 6-histidine tagged granzyme B. Forprotein expression, sf9 cells were grown to a density of 1.5×10⁶ cellsml⁻¹ in Grace's medium (Cat. 11605-094) supplemented with 10% fetal calfserum and penicillin-streptomycin-glutamine (Cat. 10378-024). Prior tothe addition of virus, cells were centrifuged at 1000×g for 15 minutesand resuspended to the same density in fresh growth medium containingrecombinant viral stock and SP Sepharose beads (4 ml resin per liter ofculture). After 72 hours of induction at 27° C., the resin was collectedin a 50 micron Nylon mesh (PGC Scientifics), extensively washed with 50mM Mes.NaOH, 0.3 M Na Cl, pH 6.6 and poured onto a column. The proteinwas eluted using a linear gradient (0.3 to 1.0 M) of NaCl. Fractionscontaining granzyme B activity were identified, combined andconcentrated and then diluted in 20 mM Tris.HCl, pH 8.0. The sample wasreadjusted to a final pH 8.0, CaCl₂ (3 mM final concentration) andfactor Xa (Pharmacia, 10 units per mg of protein) were added, andincubated for 18 hours at room temperature to generate active granzymeB. Under these conditions, complete cleavage is achieved. Therecombinant granzyme B was purified from the cleavage mixture using a 1ml HiTrap SP column (Pharmacia). The yield can be as much as 4-5 mg ofpurified granzyme B per liter of culture. Mass spectral analysisidentifies one major peak at 27,466 Da (other components of 27,320 Daand 26,630 Da were also consistently observed). Since the combined massof the 227-amino acids defined by sequence analysis only accounts for25,511.58 Da, we concluded that preparations of recombinant granzyme Bpurified with this method are more homogenous and less glycosylated thanpreparations of native granzyme B purified from NK cell granules.Nevertheless, the resulting enzyme is indistinguishable from nativeenzyme with regard to kinetic parameters for inhibition by Ac-IEPD-CHOand other inhibitors.

Fluorogenic Assay for Granzyme B

The activity of granzyme B was measured using a continuous fluorometricassay using the substrate Ac-IEPD-AMC. Briefly, appropriate dilutions ofenzyme were added to reaction mixtures containing substrate (10 μM), andvarious concentrations of the inhibitor of interest in a final reactionvolume of 100 μl. Liberation of AMC was monitored continuously at roomtemperature using a Tecan Fluostar 96-well plate reader (black platesfrom Dynatech) using an excitation wavelength of 380 nm and an emissionwavelength of 460 n m. Unless otherwise indicated, all experiments werecarried out at room temperature under standard reaction conditionsdefined as 0.1 M HEPES, 10% sucrose, 0.1% CHAPS and 10 mM DTT, pH=7.5,25° C.

Data Analysis

All kinetic constants were computed by direct fits of the data to theappropriate equation using a nonlinear least squares analysis computerprogram (NLIN). Reaction rates were fit by non-linear regression to theMichaelis-Menton equation for competitive inhibition to obtain a valuefor the dissociation constant K_(i). In cases where inhibition wastime-dependent, K_(i) was instead calculated from the steady-statevelocities, or from the rate constants for association (k_(on)) anddissociation (k_(off)) of enzyme-inhibitor complex according to theequations developed by Morrison for analysis of slow and tight-bindinginhibitors (21). The errors in reproducing the rate and dissociationconstants were never more than 10%.

Biological Data

The table below shows activity of selected examples. All compounds wereinactive against caspase enzymes.

EXAMPLE # R₁ R₂ Gzm B K_(i) (nM) 1

74 2

38 3

75 4

13 19

788 21

410 23

22 24

36 25

203 26

17 27

45 28

7 29

3880 30

112 31

1841 32

1021 33

71Measurement of Caspase Activity by Cleavage of a Fluorogenic Substrate

Assays for measuring the activity of caspases are well known in the art.For example, the following procedure can be followed to determine theactivity of caspase-3:

A fluorogenic derivative of the tetrapeptide recognized by caspase-3 andcorresponding to the P₁ to P₄ amino acids of the PARP cleavage site,Ac-DEVD-AMC (AMC amino-4-methylcoumarin) was prepared as follows: i)synthesis of N-Ac-Asp(OBn)-Glu(OBn)-Val-CO₂H, ii) coupling withAsp(OBn)-7-amino-4-methylcoumarin, iii) removal of benzyl groups.

Standard reaction mixtures (300 μL final volume), contained Ac-DEVD-AMCand purified or crude caspase-3 enzyme in 50 mM Hepes/KOH (pH 7.0), 10%(v/v) glycerol, 0.1% (w/v) CHAPS, 2 mM EDTA, 5 mM dithiothreitol, andwere incubated at 25° C. Reactions were monitored continuously in aspectrofluorometer at an excitation wavelength of 380 nm and an emissionwavelength of 460 nm.

Assays for determining the activity of the other caspases are easilydiscernible by those skilled in the art.

Data analysis may be performed as described above.

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1. A compound represented by Formula I:

or a pharmaceutically acceptable salt or hydrate thereof, wherein: n is0, 1, or 2; R¹ and R² are each independently selected from the groupconsisting of: hydrogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₃₋₆cycloalkyl, aryl,HET and —N(R¹⁰)₂, wherein: (a) said C₁₋₆alkyl, C₁₋₆alkoxy andC₃₋₆cycloalkyl are optionally substituted with 1-3 substituentsindependently selected from the group consisting of halo and hydroxy;and (b) said aryl and BET are optionally substituted with 1-3substituents independently selected from the group consisting of: halo,hydroxy and C₁₋₄alkyl, optionally substituted with 1-3 halo groups; orR1 and R2 may be joined together with the carbon atom to which they areattached to form a five or six membered monocyclic ring, optionallycontaining 1-3 heteroatoms selected from the group consisting of: S, Oand N(R¹⁰), wherein said ring is optionally substituted with 1-3 R¹⁰groups, with the proviso that R¹ and R² are both not hydrogen; each ofR³ and R⁷ is independently selected from the group consisting of:hydrogen and C₁₋₄allyl, optionally substituted with 1-3 halo groups;each of R⁴, R⁵, R⁶ and R⁸ is independently selected from the groupconsisting of: hydrogen, halo, hydroxy and C₁₋₄alkyl, optionallysubstituted with 1-3 halo groups; R⁹ is HET, optionally substituted with1-3 substituents independently selected from the group consisting of:halo, hydroxy and C₁₋₄allyl, optionally substituted with 1-3 halogroups; R¹⁰ is selected from the group consisting of: hydrogen,C₁₋₄alkyl and —C(O)C₁₋₄alkyl, said —C(O)C₁₋₄alkyl optionally substitutedwith N(R¹¹)₂, HET and aryl, said aryl optionally substituted with 1-3halo groups; R¹¹ is selected from hydrogen and C₁₋₄alkyl, optionallysubstituted with 1-3 halo groups; HET is a 5- to 10-membered aromatic,partially aromatic or non-aromatic mono- or bicyclic ring, containing1-4 heteroatoms selected from O, S and N(R¹²), and optionallysubstituted with 1-2 oxo groups; and R¹² is selected from the groupconsisting of: hydrogen and C₁₋₄alkyl, optionally substituted with 1-3halo groups.
 2. The compound according to claim 1 wherein n is
 0. 3. Thecompound according to claim 1 wherein n is
 1. 4. The compound accordingto claim 1 wherein n is
 2. 5. The compound according to claim 1 whereineach of R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is hydrogen.
 6. The compound accordingto claim 1 wherein HET is selected from the group consisting of:benzimidazolyl, benzofuranyl, benzopyrazolyl, benzotriazolyl,benzothiophenyl, benzoxazolyl, cinnolinyl, furanyl, imidazolyl,indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxadiazolyl,oxazolyl, pyrazinyl, pyrazolyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, tetrazolyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, tetrahydrofuranyl, andtetrahydrothienyl, each optionally substituted with 1-2 substituentsindependently selected from the group consisting of: halo, oxo, hydroxyand C₁₋₄alkyl, optionally substituted with 1-3 halo groups.
 7. Thecompound according to claim 1 wherein R⁹ is selected from the groupconsisting of: pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, imidazolyl,pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl,1,2,4-triazolyl, 1,2,3-triazolyl, and tetrazolyl, each optionallysubstituted with 1-3 substituents independently selected from the groupconsisting of: halo, hydroxy and C₁₋₄alkyl, optionally substituted with1-3 halo groups.
 8. The compound according to claim 1 wherein R¹ and R²are each independently selected from the group consisting of: C₁₋₆alkyl,C₃₋₆cycloalkyl, phenyl, pyridyl, 2-oxopyrrolidine and —N(R¹⁰)₂, wherein:(a) said C₁₋₆alkyl and C₃₋₆cycloalkyl optionally substituted with 1-3groups independently selected from the group consisting of halo andhydroxy; and (b) said phenyl, pyridyl and 2-oxopyrrolidine optionallysubstituted with 1-3 groups independently selected from the groupconsisting of: halo, hydroxy and C₁₋₄alkyl, optionally substituted with1-3 halo groups; and R¹⁰ is selected from the group consisting of:hydrogen, C₁₋₄alkyl, and —C(O)C₁₋₄alkyl, said —C(O)C₁₋₄alkyl optionallysubstituted with N(R¹¹)₂, pyrrolidine, piperidine, morhpoline,benzothiophene and phenyl, said phenyl optionally substituted with 1-3halo groups.
 9. The compound according to claim 8 wherein n is
 1. 10.The compound according to claim 9 wherein each of R³, R⁴, R⁵, R⁶, R⁷ andR⁸ is hydrogen.
 11. The compound according to claim 10 wherein R⁹ isselected from the group consisting of: pyridyl, pyrimidyl, pyrazinyl,pyridazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl,isoxazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, and tetrazolyl, eachoptionally substituted with 1-3 substituents independently selected fromthe group consisting of: halo, hydroxy and C₁₋₄alkyl, optionallysubstituted with 1-3 halo groups.
 12. The compound according to claim 1of Formula II:

or a pharmaceutically acceptable salt or hydrate thereof, wherein: R⁹ isselected from the group consisting of: benzimidazolyl, benzofuranyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl,indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl,pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, tetrazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, tetrahydrofuranyl, andtetrahydrothienyl.
 13. The compound according to claim 12 wherein R⁹ isselected from the group consisting of: pyridyl, pyrimidyl, pyrazinyl,pyridazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl,isoxazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, and tetrazolyl.
 14. Acompound selected from the group consisting of:

A B


15. A pharmaceutical composition comprising a compound in accordancewith claim 1 in combination with a pharmaceutically acceptable carrier.16. A method of treating an immunoregulatory abnormality in a mammalianpatient in need of such treatment comprising administering to saidpatient a compound in accordance with claim 1 in an amount that iseffective for treating said immunoregulatory abnormality.
 17. The methodaccording to claim 16 wherein the immunoregulatory abnormality is anautoimmune or chronic inflammatory disease selected from the groupconsisting of: systemic lupus erythematosis, chronic rheumatoidarthritis, type I diabetes mellitus, inflammatory bowel disease, biliarycirrhosis, uveitis, multiple sclerosis, Crohn's disease, ulcerativecolitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmunemyositis, Wegener's granulomatosis, ichthyosis, Graves ophthalmopathy,asthma, schleroderma and Sjogren's syndrome.
 18. The method according toclaim 16 wherein the immunoregulatory abnormality is bone marrow ororgan transplant rejection or graft-versus-host disease.
 19. The methodaccording to claim 16 wherein the immunoregulatory abnormality isselected from the group consisting of: transplantation of organs ortissue, graft-versus-host diseases brought about by transplantation,autoimmune syndromes including rheumatoid arthritis, systemic lupuserythematosus, Hashimoto's thyroiditis, multiple sclerosis, myastheniagravis, type I diabetes, uveitis, posterior uveitis, allergicencephalomyelitis, glomerulonephritis, post-infectious autoimmunediseases including rheumatic fever and post-infectiousglomerulonephritis, inflammatory and hyperproliferative skin diseases,psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis,seborrhoeic dermatitis, lichen planus, pemphigus, bullous pemphigoid,epidermolysis bullosa, urticaria, angioedemas, vasculitis, erythema,cutaneous eosinophilia, lupus erythematosus, acne, alopecia areata,keratoconjunctivitis, vernal conjunctivitis, uveitis associated withBehcet's disease, keratitis, herpetic keratitis, conical cornea,dystrophia epithelialis corneae, corneal leukoma, ocular pemphigus,Mooren's ulcer, scleritis, Graves' opthalmopathy, Vogt-Koyanagi-Haradasyndrome, sarcoidosis, pollen allergies, reversible obstructive airwaydisease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsicasthma, dust asthma, chronic or inveterate asthma, late asthma andairway hyper-responsiveness, bronchitis, gastric ulcers, vascular damagecaused by ischemic diseases and thrombosis, ischemic bowel diseases,inflammatory bowel diseases, necrotizing enterocolitis, intestinallesions associated with thermal burns, coeliac diseases, proctitis,eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerativecolitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischemic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, chronic bacterialinfection, malignancy of lymphoid origin and acute and chroniclymphocytic leukemias and lymphomas.
 20. A method of suppressing theimmune system in a mammalian patient in need of immunosuppressioncomprising administering to said patient an immunosuppressing effectiveamount of a compound of claim
 1. 21. A pharmaceutical compositioncomprising a compound which inhibits granzme B and does notsubstantially inhibit any caspase protease in combination with apharmaceutically acceptable carrier.
 22. The pharmaceutical compositionin accordance with claim 21 comprising a compound which possesses a Kiof 500 nM or less for inhibiting granzyme B and possesses a Ki of 10,000nM or more for inhibiting each of caspase-1 to caspase-13 in combinationwith a pharmaceutically acceptable carrier.
 23. A method of treating animmunoregulatory abnormality in a mammalian patient in need of suchtreatment comprising administering to said patient a compound whichinhibits granzme B and does not substantially inhibit any caspaseprotease in an amount that is effective for treating saidimmunoregulatory abnormality.
 24. The method of treating animmunoregulatory abnormality in a mammalian patient in need of suchtreatment in accordance with claim 23 comprising administering to saidpatient a compound which possesses a Ki of 500 nM or less for inhibitinggranzyme B and possesses a Ki of 10,000 nM or more for inhibiting eachof caspase-1 to caspase-13 in an amount that is effective for treatingsaid immunoregulatory abnormality.